wnt signaling and drug resistance in...

1521-0111/97/2/72–89$35.00 https://doi.org/10.1124/mol.119.117978MOLECULAR PHARMACOLOGY Mol Pharmacol 97:72–89, February 2020Copyright ª 2020 by The Author(s)This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

Special Section on New Opportunities in Targeting WNT Signaling – Minireview

Wnt Signaling and Drug Resistance in Cancer

Zheng Zhong and David M. VirshupDepartment of Physiology, National University of Singapore, Singapore, Singapore (Z.Z.); Program in Cancer and Stem CellBiology, Duke-NUS Medical School, Singapore, Singapore (Z.Z., D.M.V.); and Department of Pediatrics, Duke University,Durham, North Carolina (D.M.V.)

Received September 17, 2019; accepted November 21, 2019

ABSTRACTWnts are secreted proteins that bind to cell surface receptors toactivate downstream signaling cascades. Normal Wnt signalingplays key roles in embryonic development and adult tissuehomeostasis. The secretion of Wnt ligands, the turnover of Wntreceptors, and the signaling transduction are tightly regulatedand fine-tuned to keep the signaling output “just right.” Hyper-activated Wnt signaling due to recurrent genetic alterationsdrives several human cancers. Elevated Wnt signaling alsoconfers resistance to multiple conventional and targetedcancer therapies through diverse mechanisms including maintain-ing the cancer stem cell population, enhancing DNA damagerepair, facilitating transcriptional plasticity, and promoting immuneevasion. Different classes of Wnt signaling inhibitors targeting keynodes of the pathway have been developed and show efficacy in

treating Wnt-driven cancers and subverting Wnt-mediated ther-apy resistance in preclinical studies. Several of these inhibitorshave advanced to clinical trials, both singly and in combinationwith other existing US Food and Drug Administration–approvedanti-cancer modalities. In the near future, pharmacologicalinhibition of Wnt signaling may be a real choice for patientswith cancer.

SIGNIFICANCE STATEMENTThe latest insights inWnt signaling, ranging from basic biology totherapeutic implications in cancer, are reviewed. Recent studiesextend understanding of this ancient signaling pathway anddescribe the development and improvement of anti-Wnt ther-apeutic modalities for cancer.

IntroductionWnt signaling is an evolutionarily conserved signaling trans-

duction pathway that plays important roles in embryonicdevelopment and adult tissue homeostasis. DysregulatedWnt signaling causes human cancers, and an increasingnumber of studies reveal that elevated Wnt signaling contrib-utes to drug resistance in cancer therapy. Given the core role ofWnt signaling in multiple cancers, the Wnt pathway has beenone of the hottest targets of drug development. Several Wntpathway inhibitors have been developed and show promisingefficacy in treating Wnt-driven cancers and subverting

Wnt-mediated therapy resistance in preclinical studies. Inthis review, wewill present an update on recent findings in theWnt signaling transduction pathway and advances in phar-macological targeting of Wnt signaling. We will focus on theroles of Wnt signaling in cancer drug resistance as well asanti-Wnt signaling-based combination therapies. As multipleWnt inhibitors are being widely used in preclinical studies andsome have advanced to clinical trials, resistance toWnt blockadeis also observed in certain Wnt-dependent normal tissue andcancers. Here we will also summarize the potential mecha-nisms that confer resistance to Wnt inhibitors.

Wnt Signaling PathwayWnt Genes and Proteins. Wnts are secreted proteins

encoded byWnt genes that are present in all clades ofmetazoans(Holstein, 2012). The first mammalian Wnt gene, mouse Int-1,

This study is supported in part by the National Research Foundation Singaporeand administered by the Singapore Ministry of Health’s National MedicalResearch Council under the Singapore Translational Research (STaR) AwardProgram to D.M.V. Z.Z. was supported by the National University of Singapore(NUS) Research Scholarship. D.M.V. has a financial interest in ETC-159.

https://doi.org/10.1124/mol.119.117978.

ABBREVIATIONS: AA/P, abiraterone acetate/prednisone; ABC, ATP-binding cassette; AKT, Ak strain transforming; APC, adenomatouspolyposis coli; AXIN, axis inhibition protein; BCC, basal cell carcinoma; BET, bromodomain and extra terminal protein; BRAF, V-Raf murinesarcoma viral oncogene homolog B; CK1a, casein kinase 1 alpha; CRD, cysteine-rich domain; EGFR, epidermal growth factor receptor; ER,endoplasmic reticulum; EGFR, epidermal growth factor receptor; FOXO, O subclass of the Forkhead family; Fzd, Frizzled; GSK3, glycogen synthasekinase 3; iCRT, inhibitor of b-catenin responsive transcription; MAPK, mitogen-activated protein kinase; MDR, multidrug resistance; MMTV, mousemammary tumor virus; MYC, V-Myc Avian Myelocytomatosis viral oncogene homolog; PARP, poly (ADP-ribose) polymerase; PDGFR, platelet-derived growth factor receptor; PORCN, porcupine; RNF43, ring finger protein 43; RSPO, R-Spondin; TCF/LEF, T-cell factor/lymphoid enhancer-binding factor; USP6, ubiquitin-specific protease 6; Wnt, Wingless and Int; ZNRF3, zinc and ring finger 3.

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was identified in 1982 as a proto-oncogene in the mouse genomewhose expressionwasactivated bymousemammary tumorvirus(MMTV) proviral DNA integration leading to mouse mam-mary cancers (Nusse and Varmus, 1982). The Int-1 gene wassubsequently found to be a homolog of theDrosophilaWinglessgene that controls segment polarity in fly larval development(Rijsewijk et al., 1987). Therefore, genes of this family are calledWnt as a blend, or portmanteau, ofWingless and Int (Nusse et al.,1991). There are 19 Wnt genes in most mammalian genomes,including the human genome, which can be categorized into12 subfamilies.Wnt proteins are 350–400 amino acids in length and

∼40 kDa in size. They contain 22 or 24 conserved cysteineresidues that form 11 or 12 intramolecular disulfide bondsthat are important for the proper folding of the peptides intofunctionally activeproteins (Macdonald et al., 2014).Wntproteinsundergo post-translational modification after translation.Besides glycosylation, allWnt proteins require a post-translationalmodification, the addition of a palmitoleate moiety on a conservedserine residue, catalyzed by a membrane-bound O-acyltransferasecalled PORCN in the endoplasmic reticulum (ER) (Takadaet al., 2006; Najdi et al., 2012; Proffitt andVirshup, 2012). Thisserine residue is conserved in all Wnts across different speciesexcept in a distantly related Wnt gene named WntD inDrosophila that regulates NF-kb rather than b-catenin sig-naling (Fig. 1, A andB) (Ching et al., 2008). The palmitoleationis necessary for the interaction of Wnts with a cargo receptorcalled WLS that transports Wnts from ER to the plasmamembrane (Coombs et al., 2010; Yu et al., 2014). The palmito-leic moiety is also required for direct binding sites for Wntligand and its receptor Frizzled on the cell membrane (Fig. 1, Cand D) (Janda et al., 2012; Hirai et al., 2019). Previous studiesfrom our group have shown that all assessable human Wntslost signaling activity whenPORCNwas knocked out, and thiscould be rescued by PORCN re-expression (Najdi et al., 2012).Because of the important role of PORCN in Wnt signaling,mutations of the X-linked PORCN gene lead to embryoniclethality in males, and focal defects due to random Xinactivation in surviving females (Wang et al., 2007). TheO-linked palmitoleate makes Wnt proteins hydrophobic andtherefore limits the biologic activity of secreted Wnt proteinsto a short range, unlike circulating protein hormones such asinsulin that act at a distance.Wnt Receptors. As secreted but hydrophobic morpho-

gens, Wnts travel locally to bind to cell surface receptors andcoreceptors to activate downstream signaling transductioncascades. Wnts have several types of receptors. The bestknown are the 10 Frizzleds (Fzds) and their coreceptorsLRP5/6. However, an increasing number of coreceptors suchas GPR124, Reck, and TMEM59 (Zhou and Nathans, 2014;Posokhova et al., 2015; Vanhollebeke et al., 2015; Cho et al.,2017; Gerlach et al., 2018), and alternative receptors includingRor and Ryk have been identified (Niehrs, 2012). The Frizzledreceptors are seven-transmembrane proteins and haveN-terminalextracellular cysteine-rich domains (CRDs). The crystal structureof XenopusWNT8 in complex with mouse Frizzled-8 CRD revealsthe multiple interacting surfaces of Wnt-Fzd binding, includinga hydrophobic groove in the CRD that binds to the hydrophobicpalmitoleate on Wnt (Janda et al., 2012). Additional struc-tures have extended these results, suggesting palmitoleatebinding serves to dimerize Frizzled CRDs (Hirai et al., 2019;Nile and Hannoush, 2019).

The abundance of cell surface Frizzled proteins is regulatedby post-translational modification. Recent studies identifiedtwo highly homologous Wnt target genes called RNF43 andZNRF3, encoding two transmembrane RINGdomain-containingE3 ubiquitin ligases. RNF43 and ZNRF3 can ubiquitinate thecytosolic domain of Frizzleds causing the internalization anddegradation of Frizzleds. As Wnt target genes, active Wntsignaling upregulates the expression of RNF43 and ZNRF3,which in turn downregulate the surface Wnt receptors leveland the downstreamWnt signaling in a negative feedback loop(Fig. 2, A and B) (Hao et al., 2012; Koo et al., 2012). ThisRNF43/ZNRF3 mediated membrane clearance of Wnt recep-tors is tightly regulated by at least two processes. First, theubiquitin-specific protease 6 (USP6) reverses the effects ofRNF43/ZNRF3 by deubiquitinating Frizzleds (Fig. 2C) (Madanet al., 2016b). Second, R-Spondins (RSPO1-4) are secretedprotein ligands that bind to the extracellular domains of bothRNF43/ZNRF3 and LGR4/5 and lead to their clearance fromthe membrane (Fig. 2D) (Niehrs, 2012; de Lau et al., 2014).The Wnt/b-Catenin Signaling Cascade. Binding of

Wnt ligands to their various receptors can activate differentdownstream signaling pathways. The Wnt/b-catenin signaltransduction (canonical Wnt pathway) is the most well-studiedWnt pathway. b-catenin, encoded by the CTNNB1 gene, playsa central role in this pathway. b-catenin is a dual functionprotein, involved in both cell-cell adhesion and regulation ofgene transcription. At the cell membrane, b-catenin is part ofa protein complex that forms the adherens junction, which isfundamental for the maintenance of the epithelial cell layers.Nuclear b-catenin acts as transcriptional regulator. The abun-dance of b-catenin is tightly regulated by a destruction complexthat contains the scaffold protein AXIN, APC (encoded by thewell-known tumor suppressor gene adenomatous polyposis coli[APC]), casein kinase 1 alpha (CK1a), and glycogen synthasekinase 3 alpha and beta (GSK3a/b) (Asuni et al., 2006; Dobleet al., 2007; Macdonald et al., 2009).In the absence ofWnt ligands (Fig. 3, left panel), b-catenin is

first phosphorylated by CK1a at Ser45, and subsequentlyphosphorylated by GSK3a/b at Thr41, Ser37, and Ser33 (Amitet al., 2002; Liu et al., 2002). The F-box protein, b-transducinrepeats containing protein recognizes and binds to this phos-phorylated b-catenin, mediating its ubiquitination by the Skp1,Cullin, F-box E3 ubiquitin ligase complex and subsequentproteasomal degradation (Jiang and Struhl, 1998; Hart et al.,1999; Liu et al., 1999; Winston et al., 1999).In the presence ofWnt ligands (Fig. 3, right panel), Frizzleds

and the coreceptors LRP5/6 multimerize at the cell surface.This leads to recruitment of the cytoplasmic protein Dish-evelled to the cell membrane by interacting with cytoplasmicdomains of Frizzleds (Wong et al., 2003; Ma et al., 2019). TheFrizzled-boundDishevelled recruits the AXIN complex throughthe DIX domains on Dishevelled and AXIN (Schwarz-Romondet al., 2007). GSK3a/b in the AXIN complex initiates phosphor-ylation of the PPPSPmotifs of the LRP5/6 cytoplasmic tail andprimes subsequent phosphorylation byCK1s (Zeng et al., 2005).As docking sites, these phosphorylated LRP5/6 PPPSP motifsrecruitmoreAXIN complexes to the cellmembrane that furtherphosphorylate the LRP5/6 PPPSPmotifs as a positive feedbackloop (Zeng et al., 2008). Collectively, stimulation of Wntligands relocalizes the b-catenin destruction complex to thecell membrane. However, the exact mechanism by whichthis causes b-catenin stabilization remains controversial.

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Fig. 1. Wnts are secreted proteins with conserved domains and residues. (A) The consensus modeling of 19 humanWnts. The amino acid sequences of 19human Wnts were aligned, and the amino acid conservation scores were calculated using The ConSurf Server website (http://consurf.tau.ac.il). Theconservation scores were then mapped on the human Wnt3 crystal structure (PDB 6AHY chain B). (B) The consensus modeling of Wnt homologs. Thepalmitoleation site and Frizzled interaction sites are conserved in all Wnts. 2635 amino acid sequences that are homologs of humanWnt3 were collectedfrom UNIREF90 using the HMMER algorithm. The conservation scores were calculated from 150 amino acid sequences representative of the 2635sequences using TheConSurf Server website andmapped on the humanWnt3 crystal structure. (C) The crystal structure of humanWnt3 in complex withmouse Frizzled 8 CRD (PDB 6AHY). (D) The Wnt secretion pathway. Wnts are palmitoleated by PORCN in the ER. The palmitoleic moiety (red line)facilitates the interaction of Wnts with the cargo receptor WLS that transports Wnts to the plasma membrane. Multiple routes of Wnt release andextracellular transport including diffusion, exovesicles, and cytoneme-mediated transport have been proposed.

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Diverse models have been proposed with supporting evidence,including 1) inhibition of GSK3 activity by the phosphorylatedPPPSP motif that binds to the GSK3 catalytic pocket as a“pseudo substrate” (Mi et al., 2006; Cselenyi et al., 2008; Piaoet al., 2008; Wu et al., 2009), 2) sequestration of GSK3 fromthe cytoplasm intomultivesicular bodies (MVBs) that restrictsGSK39s access to substrates (Taelman et al., 2010), 3) dissociationof the ubiquitination machinery from the b-catenin destructioncomplex (Li et al., 2012), and 4) degradation of AXIN anddisassembly of the destruction complex (Tolwinski et al., 2003).Subsequently, the newly synthesized b-catenin protein canaccumulate in the cytoplasm and enter the nucleus to regulategene transcription. Notably, inhibition of AXIN-bound GSK3 inWnt signaling is independent of the well-studied AKT-mediatedphosphorylation on the Ser21 of GSK3a and Ser9 of GSK3b thatinhibits their kinase activity (Ding et al., 2000; McManus et al.,2005; Ng et al., 2009), and inhibition of GSK3 byWnts also blocksdegradation of other GSK3 targets including MYC (Taelmanet al., 2010; Madan et al., 2018).b-Catenin regulates gene expression in large part in

a TCF-dependent manner (Schuijers et al., 2014). The TCF/LEFfamily is a group of DNA-bound transcription factors withcognate binding motif 59-AGATCAAAGG-39. They recruitGroucho family transcriptional repressors to inhibit genetranscription in the absence of b-catenin. WhenWnt is present,b-catenin is stabilized and enters the nucleus, binds to TCF,and converts TCF into a transcriptional activator. However,there is a subset of b-catenin transcriptional targets that donot require TCF/LEF factors for their regulation. In these loci,

b-catenin interacts with other factors such as MyoD andFOXO to regulate gene transcription (Fig. 3, right panel)(Doumpas et al., 2019).The direct Wnt target genes are defined as genes with the

conserved TCF binding sites that functionally influence genetranscription. Most Wnt target genes are cell type and develop-mental stage specific, indicating their promoters are subject toadditional tissue-specific regulation (Nakamura et al., 2016).Many of theseWnt/b-catenin targets such asMYC and cyclinsregulate core biologic processes such as ribosome biogene-sis and cell cycle progression (Madan et al., 2018). It is alsointeresting that a number of the more robustWnt target genesare themselves negative regulators of Wnt signaling. Theseinclude AXIN2, RNF43, ZNRF3, and NOTUM. As describedpreviously, RNF43/ZNRF3 negatively regulate the Frizzledslevel on the cell surface (Fig. 2, A and B) (Hao et al., 2012; Kooet al., 2012). NOTUM is a palmitoleoyl-protein carboxylester-ase that can remove the palmitoleic group on Wnt ligands(Kakugawa et al., 2015). As this palmitoleic group is necessaryfor the interaction of Wnt ligand and receptor, Notum inacti-vates the function of Wnt ligands. These multiple negativefeedback mechanisms guarantee that the Wnt signaling is pre-cisely regulated and kept at the just right level.b-Catenin Independent Wnt Signaling Pathways. b-

Catenin–independent Wnt signaling is defined as Wnt-or Frizzled-initiated signaling that is independent ofTCF/b-catenin–mediated transcription. These “noncanonical”Wnt signaling pathways are diverse and their role inmammalsis less well understood. SomeWnts such as Wnt5a and Frizzleds

Fig. 2. The abundance of cell surface Wnt receptors is tightly regulated by RNF43/ZNRF3, USP6, and R-Spondins. (A) RNF43 and ZNRF3 areWnt/b-catenin target genes. (B) RNF43 and ZNRF3 ubiquitinate the cytosolic domain of Frizzleds causing the internalization and degradation ofFrizzleds. (C) USP6 reverses the effects of RNF43/ZNRF3 by deubiquitinating Frizzleds. (D) R-Spondins bind to the extracellular domains of bothRNF43/ZNRF3 and LGR4/5 leading to their membrane clearance. Ub, ubiquitin.


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are involved in the regulation of intracellular calcium levels andplanar cell polarity (Kikuchi et al., 2012). Some Wnts can bindto receptor tyrosine kinases such as ROR2 and RYK to activatedownstream signaling leading to, e.g., cytoneme extension(Mattes et al., 2018). Notably, one established effect of Wnt5ais antagonism of the canonical Wnt/b-catenin signaling (Ishitaniet al., 2003; Topol et al., 2003; Mikels and Nusse, 2006). Thespecific signaling pathway regulated by individual Wnt ligand isalso based on the specific receptors existing on the cell surfaceand is cell type and developmental stage dependent (Semenovet al., 2007; Najdi et al., 2012).Roles of Wnt Signaling in Development and Adult

Tissue. The Wnt signaling pathway is evolutionarily con-served across various species from sponges to fruit flies tohuman beings (Loh et al., 2016). As shown by both naturallyoccurring and experimentally induced loss-of-function andgain-of-function mutations in many species, Wnt signalingplays important roles in both embryonic development and inmaintaining adult tissue homeostasis.The first Wnt gene, wingless, was identified in Drosophila.

As the name suggests, the first mutant of wingless genedisplayed a phenotype of transformation of wing structuresinto thoracic notal structures. It was also first found inDrosophila that Wnt is critical for the establishment ofanterior-posterior polarity during embryonic segmentation(Swarup and Verheyen, 2012). In vertebrates, Wnt signalingalso plays a key role in controlling body axis formation. Wntsignaling can induce secondary dorsal structures when mis-activated in early Xenopus embryos. This axis duplicationassay has been widely applied to functionally test a genecandidate’s effect on the b-catenin–dependent Wnt signalingpathway. In mice, genetic studies have shown that Wnt

signaling is required for gastrulation, and Wnt3, Lrp5/6,and b-catenin knockouts generated similar phenotypes(Wang et al., 2012). In summary, Wnt signaling controlsaxis patterning, cell fate specification, cell proliferation,and cell migration in embryonic development.In healthy adult tissue, one of the most important roles

of Wnt signaling is to maintain the adult stem cells. Usinglineage tracing strategies, the Wnt target gene Lgr5 has beendemonstrated to mark stem cells, first in the intestine (Barkeret al., 2007) and then in several other tissues includingstomach, pancreas, liver, kidney, ovarian epithelium, mam-mary gland, hair follicle, inner ear, and taste buds (Leunget al., 2018).The intestine is one of the best-studied examples of Wnt

signaling and stem cell maintenance. The small intestinalepithelial layer is one of the most continuously proliferativetissues in the body and undergoes self-renewal every 3–5 days.It has a crypt-villus structure (Clevers, 2013). Villi are protru-sions of the simple columnar epithelium that extend into thegut lumen. Crypts are invaginations of the epithelium thatsurround the base of each villus. One set of intestinal stem cells(also called crypt base columnar cells) are located at the base ofthe crypt, surrounded by the Paneth cells. These stem cells thengive rise to the transit amplifying cells, which are rapidly dividingcommitted progenitor cells that differentiate into all the maturecell types of the epithelium. Themature differentiated cells moveupward from the crypt region to the villus and are finally shed aspart of the self-renewal process of the intestinal epithelium.As mentioned previously, the Wnt/b-catenin target gene

Lgr5 is exclusively expressed in the crypt base columnar cells,indicating active Wnt signaling in the intestinal stem cells.As a coreceptor for the RSPOs, LGR5 helps to sequester

Fig. 3. The canonical Wnt/b-catenin sig-naling cascade. (Left panel) In the absenceof Wnt ligands, b-catenin is phosphory-lated, ubiquitinated, and degraded by theb-catenin destruction complex. (Right panel)Binding of Wnt ligands to the receptorsrelocalizes the destruction complex to themembrane and interferes with its activity.Subsequently, newly synthesized b-cateninaccumulates in the cytoplasm and entersthe nucleus to regulate gene transcription.Refer to the text for detailed description.DVL, dishevelled.


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RNF43/ZNRF3 and upregulates the cell surface Frizzled/LRPlevels (Fig. 2D). This may work as a positive feedback loop tofurther enhance the Wnt signaling in these stem cells. Thereare additional diverse Wnt ligands expressed in the crypt-villus axis that may help regulate differentiation into othercell types (Gregorieff et al., 2005). Paneth cells reside adjacentto the intestinal stem cells at the base of the crypt and areknown for expressing several Wnts, including Wnt3. Wntssecreted by Paneth cells can support the ex vivo organoidgrowth from purified intestinal epithelial cells (Sato et al.,2011). However, one study from our group showed that micelacking epithelial Wnt activity by genetic ablation of Porcn inthe intestinal epithelium had normal intestinal homeostasisas well as normal intestinal regeneration capability afterradiation injury, indicating that epithelial Wnts (includingWnts secreted by Paneth cells) are dispensable for theseprocesses (Kabiri et al., 2014). Recently, our group identifiedthe PDGFRa1 pericryptal stromal cells as the critical source ofWnts and RSPO3 for the intestinal stem cells in vivo (Greiciuset al., 2018). And three other studies identified stromal cellsmarked by FOXL11 (Aoki et al., 2016; Shoshkes-Carmel et al.,2018) and GLI11 (Degirmenci et al., 2018), respectively, thatform the essentialWnt-secreting niche for the stem cells in thegut. Notably, there is significant but incomplete overlapamong the PDGFRa1, FOXL11, and GLI11 stromal cells(Greicius and Virshup, 2019).Importantly, Wnt/b-catenin signaling represses prolif-

eration of the intestinal stem cells. It is known that highWnt/b-catenin signaling, marked by nuclear b-cateninaccumulation and b-catenin target gene expression, is largelyrestricted to the intestinal stem cells in the crypt base,whereas the highly proliferative transit amplifying cellslack Wnt/b-catenin signaling. Instead, it is well establishedthat the EGFR/RAS/MAPK signaling drives active prolifera-tion in the intestine (Biteau and Jasper, 2011; Jin et al., 2015).One recent study found that Wnt inhibition by PORCN in-hibitor induced an initial burst of proliferation of the intestinalstem cells due to conversion of the stem cells into the transit-amplifying cells with a loss of stem cell self-renewal (Kabiriet al., 2018). This is driven by activation of theWnt-suppressedMAPK signaling. And this will eventually lead to intestinalcrypt ablation as the transit-amplifying cells differentiate andstem cells get depleted. Collectively, these findings indicatethat the role of Wnt signaling in the intestinal stem cell nicheis to maintain the stemness and inhibit differentiationrather than to promote the stem cell proliferation (Greiciusand Virshup, 2019).

Aberrant Wnt Signaling in Cancer andPharmacological Targeting of Wnt Signaling

Common Alterations of Wnt Pathway Components inCancer

The first mammalian Wnt gene, Int-1, was identifiedbecause its overexpression caused mouse mammary cancer(Nusse and Varmus, 1982). The first evidence that aberrantWnt signaling caused human cancer came from studies ofhereditary colorectal cancer. In 1991, three groups indepen-dently found that the APC gene is mutated in the hereditarycolon cancer syndrome, familiar adenomatous polyposis,as well as in many cases of the sporadic colorectal cancer(Groden et al., 1991; Kinzler et al., 1991; Nishisho et al., 1991).

Subsequent studies showed that loss-of-function mutations inthe APC gene lead to inappropriate stabilization of b-cateninand constitutive transcriptional activation by the b-catenin/TCFcomplex (Rubinfeld et al., 1996; Korinek et al., 1997; Morinet al., 1997). In agreement with the b-catenin phosphorylation/degradation model (Liu et al., 2002), point mutations in theN-terminal Ser/Thr phosphorylation sites of b-catenin thatprevent its degradation were found in a subset of colorectalcancer with wild-type APC gene (Morin et al., 1997).Aberrant activation of Wnt/b-catenin signaling is not re-

stricted to colorectal cancer. Similar alterations in b-catenin,APC, and AXIN1 are found in liver cancer cases (de La Costeet al., 1998; Anastas and Moon, 2013). A growing series ofactivating mutations in the downstream components of theWnt signaling pathway have also been reported in variousother cancer types, including cancers originating in stomach,pancreas, ovary, endometrium, kidney, adrenal gland, biliarytract, pituitary, and soft tissues (Table 1). Researchers havealso developed mouse models that confirm the tumorigeniceffect of hyperactiveWnt signaling (Colnot et al., 2004; Taketoand Edelmann, 2009; Koo et al., 2012).Mutations that activate Wnt/b-catenin signaling fall into

two broad categories: Wnt dependent and Wnt independent.Mutations inAPC,AXIN1, andCTNNB1 that stabilizeb-cateninuncouple the downstream Wnt signaling from the upstreamWnt ligands, which means that b-catenin–dependent tran-scription is constitutively activated regardless of whether theWnt ligands and receptors are present or not. More recently,studies from several groups identified activating mutationsthat areWnt dependent (Table 1). As described in the previoussection, the Wnt receptors (Frizzleds and LRPs) are tightlyregulated by two homolog E3 ubiquitin ligases (RNF43/ZNRF3)and Wnt agonists (RSPOs). As a negative feedback mechanism,highWnt signaling upregulates the expression of RNF43/ZNRF3,which leads to the membrane clearance of Wnt receptors andquenches the upstream Wnt signaling (Fig. 2, A and B).Inactivatingmutations ofRNF43 andZNRF3were first reportedin pancreatic and adrenocortical carcinomas, respectively, andsubsequently seen in cancers originating in other tissuesincluding esophagus, stomach, biliary tract, large intestine,endometrium, and ovary (Wu et al., 2011; Assié et al., 2014;Giannakis et al., 2014; Madan and Virshup, 2015). Conversely,RSPOs inhibit the activity of RNF43/ZNRF3 (Fig. 2D), andgain-of-function translocations of RSPO2 and RSPO3 wereidentified first in a subset of patients with APC wild-typecolorectal cancer (Seshagiri et al., 2012) and subsequently alsoidentified in several other cancer types (Cardona et al., 2014;Li et al., 2018; Picco et al., 2019). Either loss of function ofRNF43/ZNRF3 or gain of function of RSPO2/3 increases thesurface levels of Frizzleds and LRPs and makes cells harboringthese alterations highly sensitive to low levels of Wnt ligands.In addition, DNA methylation–mediated epigenetic silenc-

ing of genes encoding secreted frizzled-related proteins andDickkopf-related proteins that are negative regulators ofWnt signaling are common in colorectal, breast, gastric, andovarian cancers (Caldwell et al., 2004; Suzuki et al., 2004;Veeck et al., 2006, 2008; Yu et al., 2009; Zhu et al., 2012).

Targeting the Wnt Pathway in Cancer

Because of the core role Wnt signaling plays in severalhuman cancers, it has been one of the hottest targets in cancertherapeutics. Multiple inhibitors targeting the Wnt pathway


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have been developed, and a number have advanced to clinicaltrials (Table 2).Loss-of-function mutations of APC/AXIN1 or activating

mutations of b-catenin represent the most common geneticalterations that constitutively activate Wnt/b-catenin signal-ing in human cancers (Table 1). As mentioned previously,these downstreammutations uncouple b-catenin signaling fromthe upstream Wnt ligand stimulation. Therefore, blockingWnt/b-catenin signaling in these cancers requires inhibitorstargeting downstream in the pathway. In many cases of co-lorectal cancer, the truncated APC still can functionally mediateb-catenin degradation in the AXIN-containing destruction com-plex, albeit with a weakened capability (Voloshanenko et al.,2013). In this setting, an increase in AXIN protein can restoreb-catenin degradation. This can be achieved by inhibition oftankyrases TNKS1 and TNKS2, ADP ribosyltransferases thatnormally drive degradation of AXIN and other proteins (Huanget al., 2009; Riffell et al., 2012). Several tankyrase inhibitorssuch as XAV939 have been developed and show significantWnt/b-catenin signaling suppression and anticancer effect inWnt-driven cancer with APC mutations (Chen et al., 2009;Huang et al., 2009; Waaler et al., 2011, 2012; James et al.,2012; Lau et al., 2013; Okada-Iwasaki et al., 2016; Thomsonet al., 2017). Activation of CK1a in the destruction complexsimilarly causes b-catenin degradation. This was first demon-strated with pyrvinium (Thorne et al., 2010) and more recentlywith more selective molecules (Li et al., 2017).Another approach to inhibiting downstream b-catenin sig-

naling is to block the interaction of b-catenin with TCF/LEF or

associated transcriptional coactivators such as CBP/p300. Aseries of small molecule inhibitors called “inhibitor of b-cateninresponsive transcription” (iCRT) inhibits the interactionsbetween b-catenin and TCF4 and thereby suppresses thetranscriptional activity of b-catenin (Gonsalves et al., 2011).The small molecule inhibitor ICG-001 binds specifically toCBP but not the related transcriptional coactivator p300,thereby disrupting the interaction of CBP with b-catenin. Inpreclinical studies, ICG-001 can significantly suppressb-catenin/CBP–mediated transcriptional activation or repression andshows anti-tumorigenesis effects (Emami et al., 2004; Arensmanet al., 2014;Gang et al., 2014). Its active enantiomer PRI-724has entered early phase clinical trials for treating patientswith advanced tumors (Ko et al., 2016; El-Khoueiry et al.,2017). Similarly, another small molecule inhibitor calledWindorphen selectively targets the association of b-cateninwith the transcriptional coactivator p300 but not CBP (Haoet al., 2013).Upstream Inhibitors. HyperactiveWnt signaling in cancer

can also result from elevated surface Wnt receptor abundancedue to alterations in RNF43/ZNRF3 or RSPOs. To suppressWnt signaling in such cases, studies have focused on theupstream components of the Wnt pathway that include Wntligand secretion, Wnt receptor turnover, and Wnt ligand-receptor interactions. As described previously, mammalianWnts require palmitoleation by PORCN to be secreted. There-fore, small molecule inhibitors targeting PORCN can blocksecretion of all Wnts and thereby suppress the downstreamsignaling. Notably, PORCN inhibitors can shut down both

TABLE 1Common genetic alterations of Wnt pathway components in human cancers

Gene Type of Mutation Primary Tissues % Mutated Reference

APC Mainly frameshift and nonsense mutations that lead totruncated APC proteins with compromised ability todegrade b-catenin; LOH

Large intestine 70 a

Stomach 13 a

Endometrium 7 a

Liver 2.7 a

AXIN1 Mainly missense mutations, truncating mutations, and deepdeletions

Liver 7 a

Stomach 3 a

Large intestine 2.5 a

CTNNB1 Mainly missense mutations in the N-terminal Ser/Thrphosphorylation sites of b-catenin that prevent itsdegradation

Liver 29 a

Endometrium 18 a

Adrenal cortex 16 a

Large intestine 6 a

Stomach 6 a

Pancreas 2.7 a

RNF43 Mainly missense mutations and truncating mutations due toframeshift or nonsense mutations, LOH, and homozygousdeletion

Ovary (mucinous carcinoma/mucinousborderline tumor)

21/9 Ryland et al., 2013

Stomach 13 a

Biliary tract (liver fluke-associatedcholangiocarcinoma)

9.3 Ong et al., 2012

Large intestine 9 a

Pancreas 7 a

Endometrium 4 a

ZNRF3 Mainly missense mutations and truncating mutations due toframeshift or nonsense mutations, LOH, and homozygousdeletion

Adrenal cortex 20 a

Large intestine 4 a

Stomach 2.1 a

RSPO2 Chromosome rearrangement leading to the recurrent EIF3E-RSPO2 gene fusions

Large intestine 2.9 Seshagiri et al.,2012

Others (lung, head and neck,esophagus, stomach, ovary, andbreast)

1–2 Cardona et al.,2014; Li et al.,2018

RSPO3 Chromosome rearrangement leading to the recurrentPTPRK-RSPO3 gene fusions

Large intestine 7.4 Seshagiri et al.,2012

Others (lung, head and neck,esophagus, ovary, and breast)

1–11 Cardona et al.,2014

LOH, loss of heterozygosity.aCurated from the cBioPortal database (https://www.cbioportal.org) in July 2019.


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TABLE 2Inhibitors of the Wnt signaling and their effects in cancer

Targets and Functions Agent Name Functional Effects in Cancer Development Stage Reference

Small molecule tankyrase(TNKS1/2) inhibitors,stabilizing AXIN1/2

IWRs Discovery Chen et al., 2009XAV939 Inhibited colony formation of colorectal

cancer cell line DLD-1.Huang et al., 2009

WIKI4 James et al., 2012NVP-TNKS656 Shultz et al., 2013JW67, JW74 Both compounds suppressed in vitro

proliferation of colorectal cancer cellline SW480; JW74 reduced SW480in vivo tumor growth and adenomaformation in ApcMin mice.

Preclinical Waaler et al., 2011

JW55 Suppressed in vitro proliferation ofSW480; decreased adenoma formationin conditional Apc knockout mice.

Waaler et al., 2012

G007-LK Suppressed in vitro colony formation andin vivo tumor growth of colorectalcancer cell lines COLO-320DM andSW403 and spheroid formation ofApcMin mouse intestinal adenoma.

Lau et al., 2013

K-756 Suppressed in vitro proliferation ofCOLO-320DM and SW403; showeddose-dependent inhibition of Wntsignaling in DLD-1 xenografts.

Okada-Iwasaki et al., 2016

Small molecule CK1aactivators, promotingb-catenin degradation

Pyrvinium Suppressed in vitro proliferation ofcolorectal cancer cell lines SW480,DLD-1, SW620, HCT116, and HT29;reduced adenoma formation in ApcMin

mice.

Preclinical Thorne et al., 2010; Li et al.,2014;

SSTC3 Suppressed intestinal organoidformation from ApcMin mice and Apcknockout mice; reduced adenomaformation in ApcMin mice; suppressedin vitro colony formation of HT29,SW403, and HCT116 and in vivotumor growth of HCT116; suppressedtumor organoid formation from threepatient colorectal tumors and in vivogrowth of xenografts.

Li et al., 2017

Small molecule inhibitorsblocking b-catenin/TCFinteraction

iCRT3, iCRT5, andiCRT14

iCRTs led to cell cycle arrest in HCT116and HT29; iCRT3 suppressed in vitrogrowth of primary human colon cancerspecimens; iCRT14 reduced HCT116and HT29 xenograft growth.

Preclinical Gonsalves et al., 2011

Small molecule inhibitorsblocking b-catenin/CBPinteraction

ICG-001 Suppressed in vitro growth and led toapoptosis in SW480 and HCT116;suppressed in vivo growth of SW620xenografts; reduced adenomaformation in ApcMin mice; suppressedin vitro growth of pancreatic cancercell lines AsPC-1, L3.6pl, MIA PaCa-2,and PANC-1; improved survival ofAsPC-1 orthotopic xenograft-bearingmice.

Preclinical Emami et al., 2004; Arensmanet al., 2014

PRI-724 The active enantiomer of ICG-001;entered Phase 1 and 2 clinical trialsfor treating advanced cancers.

Phase 1 and 2clinical trials

ClinicalTrials.govNCT01764477,NCT01302405,NCT01606579,NCT02413853

Small molecule inhibitorblocking b-catenin/p300 interaction

Windorphen Suppressed in vitro proliferation ofcolorectal cancer cell lines SW480 andRKO and prostate cancer cell linesDU145 and PC3.

Discovery Hao et al., 2013

Small molecule PORCNinhibitors, blockingWnt secretion

IWPs Discovery Chen et al., 2009C59 Inhibited MMTV-Wnt1 tumor growth

in vivo.Preclinical Proffitt et al., 2013

LGK974 Induced regression of MMTV-WNT1tumors; inhibited in vitro colonyformation of head and neck cancer cellline HN30 and RNF43-mutantpancreatic cancer cell lines Patu8988Sand HPAF-II; inhibited in vivo tumorgrowth in xenografts of HN30 andRNF43-mutant pancreatic cancer celllines Capan2 and HPAF-II; entered

Phase 1 and 2clinical trials

Jiang et al., 2013; Liu et al.,2013; ClinicalTrials.govNCT01351103,NCT02278133,NCT02649530

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canonical and noncanonical Wnt signaling. Several PORCNinhibitors have been developed. They showed potent sup-pressive effect in preclinical models of Wnt-addicted can-cers (Jiang et al., 2013; Liu et al., 2013; Proffitt et al., 2013;Madan et al., 2016; Bhamra et al., 2017; Li et al., 2018) andhave entered clinical trials. In addition, neutralizing anti-bodies targeting Wnt receptors (Frizzleds and LRPs) andagonists (RSPOs) and soluble Fzd-based decoy receptor ofWnt ligands have been developed by different groups. Some ofthese have also entered clinical trials (Ettenberg et al., 2010;

Gurney et al., 2012; Madan and Virshup, 2015; Chartier et al.,2016; Jimeno et al., 2017; Steinhart et al., 2017).

Wnt Signaling Mediated Resistance inCancer Therapy

Wnt Signaling in Cancer Stem Cell. Stem cells aredefined as multipotent cells that can perpetuate themselvesthrough self-renewal and can also differentiate into maturecells of a particular tissue type. They are present in many

TABLE 2—Continued

Targets and Functions Agent Name Functional Effects in Cancer Development Stage Reference

Phase 1 and 2 clinical trials fortreating cancers.

ETC-159 Inhibited in vitro colony formation ofteratocarcinoma cell lines PA-1 andRNF43-mutant pancreatic andovarian cancer cell lines HPAF-II,AsPC-1, and MCAS; suppressedin vivo tumor growth in xenografts ofHPAF-II, AsPC-1, teratocarcinomacell lines PA-1 and NCCIT, andpatient-derived colorectal tumorxenografts with RSPO3 fusions;entered Phase 1 clinical trial fortreating advanced cancers.

Phase 1 clinicaltrial

Madan et al., 2016a;ClinicalTrials.govNCT02521844

RXC004 Suppressed in vivo tumor growth ofCapan2 xenografts; entered Phase 1clinical trial for treating advancedcancers.


Bhamra et al., 2017;ClinicalTrials.govNCT03447470

CGX1321 Suppressed in vivo tumor growth ofpatient-derived gastric and colorectaltumor xenografts with RSPO2 fusions;entered Phase 1 clinical trial fortreating advanced cancers.


Li et al., 2018; ClinicalTrials.gov NCT03507998,NCT02675946

Decoy Wnt receptor Ipafricept (OMP-54F28)

Suppressed in vivo tumor growth ofpatient-derived hepatocellularcarcinoma, pancreatic and ovariancancer xenografts, and decreased thecancer stem cell frequency; enteredPhase 1 clinical trials for treatingadvanced cancers; in a Phase 1 clinicaltrial, two desmoid tumor, anda patient with germ cell cancer treatedwith Ipafricept experienced stabledisease for .6 mo.

Phase 1 clinicaltrials

Jimeno et al., 2017;ClinicalTrials.govNCT02069145,NCT02092363,NCT02050178,NCT01608867

Anti-Frizzled1,2,5,7,8antibody

Vantictumab (OMP-18R5)

Suppressed in vivo tumor growth ofpatient-derived colorectal, breast,lung, and pancreatic tumor xenograftsand PA-1 xenografts; decreased thecancer stem cell frequency; enteredPhase 1 clinical trials for treatingcancers.

Phase 1 clinicaltrials

Gurney et al., 2012;ClinicalTrials.govNCT01345201,NCT02005315,NCT01957007,NCT01973309

Anti-Frizzled5,8antibodies

IgG-2919, IgG-2921 Both antibodies suppressed in vitroproliferation of RNF43-mutantpancreatic cancer cell lines HPAF-II,Patu8988S, and AsPC-1, and led tocell cycle arrest; IgG-2912 suppressedin vivo tumor growth of HPAF-II andAsPC-1 xenografts and organoidformation of RNF43-mutant colorectalcancers.

Preclinical Steinhart et al., 2017

Anti-LRP6 antibodies A7-IgG, B2-IgG A7-IgG specifically inhibited tumorgrowth of MMTV-Wnt1 xenografts;B2-IgG specifically inhibited tumorgrowth of MMTV-Wnt3 xenografts.

Preclinical Ettenberg et al., 2010

Anti-RSPO antibodies Anti-RSPO1 antibody,anti-RSPO2antibody, anti-RSPO3 antibodies

Suppressed in vivo tumor growth ofpatient-derived colorectal, lung, andovarian tumor xenografts withcorresponding RSPO overexpression;Anti-RSPO3 antibody (OMP-131R10)has entered Phase 1 clinical trial fortreating advanced solid tumors andmetastatic colorectal cancers.

Preclinical, phase1 clinical trial(OMP-131R10)

Chartier et al., 2016; Stormet al., 2016;ClinicalTrials.govNCT02482441


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adult tissues and organs and help to maintain the normaltissue architecture (Reya et al., 2001). Over the past twodecades, more and more studies have revealed the hetero-geneity within a tumor population in various cancer types.The cancer stem cell model is one way to explain the intra-tumor heterogeneity (Visvader and Lindeman, 2008). Similarto normal stem cells, cancer stem cells refer to a subset oftumor cells that undergo self-renewal while generating differ-entiated cells that comprise the bulk of the tumor. However,compared with the normal tissue stem cells, cancer stem cellsproliferate and differentiate aberrantly. Many studies haveshown that cancer stem cells are more resistant to traditionalchemotherapy and radiotherapy, which could be because ofthe higher expression of antiapoptotic proteins such as MYC,multidrug resistance genes such as the ATP-binding cassette(ABC) transporters including multidrug resistance (MDR)1 and MRP1, and more efficient DNA damage repair. There-fore, inmany cancers, although chemotherapy or radiotherapycan kill a majority of the proliferating cells leading to tumorshrinkage, the cancer stem cells can survive and lead to cancerrelapse. Cancer stem cells were also reported to contribute totumor metastasis.Wnt signaling is well-known for regulating stemness in

many normal tissues. Several studies have shown that Wntsignaling also plays important roles in maintaining cancerstem cells in various cancer types.CD341 bulge stem cells maintain follicular homeostasis in

mouse skin. In a mouse cutaneous tumor model induced bychemical carcinogens (DMBA/TPA), CD341 cells are signif-icantly enriched by 9-fold in the tumor tissues compared withthe normal skin. And the CD341 tumor cells are 100-fold morepotent in initiating tumors than unsorted tumor cells in thelimiting-dilution transplantation assay, which means thisCD341 population contains the cancer stem cells of thismouse tumor model. It was further shown that Wnt/b-cateninsignaling is essential in sustaining the CD341 cancer stemcell phenotype and that b-catenin deletion leads to the loss ofCD341 cancer stem cells as well as complete tumor regression(Malanchi et al., 2008).In pancreatic cancer, a small population of cancer cells with

high intrinsic Wnt/b-catenin signaling showed propertiesindicative of cancer stem cells, including higher tumor-initiatingcapacity and drug resistance, whereas cancer cells with low Wntactivity expressedmarkers of differentiation. In these tumors,RSPO2 was shown to enhance Wnt/b-catenin signaling toconfer stemness traits to susceptible pancreatic cancer cells(Ilmer et al., 2015).In mouse and human lung adenocarcinomas, two distinct

cell subpopulationswere identified, onewith highWnt/b-cateninsignaling and the other forming the niche that provides theWnt ligands. TheWnt responding cells expressed the stem cellmarker LGR5 and had increased tumor propagation capacity.They comprised a small minority of the tumor but could giverise to the other cell populations of the tumor bulk. The evidencesuggests that these cancer cells with active Wnt signaling havefeatures of normal tissue stem cells. Genetic and pharmacolog-ical perturbation of Wnt signaling in this model significantlysuppressed tumor progression (Tammela et al., 2017).In colorectal cancer, even thoughmost of the cancer cases have

elevatedWnt/b-catenin signaling due tomutations/alterations inthe Wnt pathway components, the Wnt signaling level reflectedby b-catenin localization and target genes expression is still very

heterogeneous within the tumor. It is reported that high Wntsignaling functionally designates the cancer stem cells incolorectal cancer. TheseWnt high tumor cells are located closeto stromal myofibroblasts and respond to the myofibroblast-secreted factors to activate b-catenin–dependent transcrip-tions. Notably, myofibroblast-secreted factors can also restorethe cancer stem cell phenotype in more differentiated tumorcells, suggesting that the dynamic stemness in colorectalcancer is not just defined by highWnt signaling, instead is alsoorchestrated by the tumor microenvironment (Vermeulenet al., 2010).Recent studies also revealed the involvement of noncoding

RNAs in regulating stemness of cancer cells. Many of thereported noncoding RNAs confer stemness traits to cancercells through activating Wnt/b-catenin signaling (Zhan et al.,2017). microRNA-146a represses Numb and stabilizes b-cateninto maintain the symmetric division of colorectal cancer stemcells (Hwang et al., 2014). In liver cancer stem cells, the longnoncoding RNA lncTCF7 is highly expressed and triggersTCF7 expression by recruiting the SWI/SNF complex to TCF7promoter, thereby activating Wnt signaling. lncTCF7 is re-quired for the self-renewal and tumorigenic capacity of livercancer stem cells (Wang et al., 2015). miR-142 promotesmRNA degradation of APC to activateWnt signaling in breastcancer stem cells (Isobe et al., 2014), whereas miR-582-3ptargets mRNAs of Wnt signaling negative regulators AXIN2,DKK3, and SRP1 for degradation to maintain stemness featuresin non–small cell lung cancer (Fang et al., 2015).Wnt Signaling Mediated Resistance to Conventional

Chemotherapy and Radiotherapy. Conventional chemo-therapy exposes patients with cancer to cytotoxic agents toblock proliferation and lead to cell death of rapidly proliferatingcells including but not limited to cancer cells. Radiotherapyapplies ionizing radiation to tumor tissues, which damagesthe DNA of cancer cells and leads to cell death. Cancer stemcells are known to be more resistant to traditional chemother-apeutic agents and radiation compared with the non–stem cellpopulations of the tumor bulk, and thereby may be able tocause cancer relapse after chemotherapy or radiotherapy. Ithas been discussed in the previous section that Wnt signalingis important in maintaining the cancer stem cells, thereforecontributing to therapeutic resistance in several cancer types.However, the protective effect conferred by Wnt signaling

against chemotherapy and radiotherapy is not restricted tocancer stem cells but also applies to the non–stem cancer cellpopulations. It has been reported in many cancers that theWnt/b-catenin activity level positively correlates with theresistance to several common chemotherapeutic drugs andradiation. Chemotherapy or radiation can upregulateWnt signal-ing, and upregulation of Wnt signaling protects cancer cells fromcell cycle arrest or apoptosis. Although this protective effect hasbeen attributed to the downstream effectors of Wnt/b-cateninsignaling such as survivin in one study, more generally theunderlining mechanism still remains unclear (Watson et al.,2010; Gao et al., 2013; Nagano et al., 2013; Emons et al., 2017).Two recent studies found that elevated Wnt signaling enhancedthe DNA damage repair and thereby conferred resistance to thePARP inhibitor olaparib in ovarian cancers (Fukumoto et al.,2019; Yamamoto et al., 2019). Thismechanism could also protectcancer cells from DNA damage caused by other conventionalchemo agents or radiation. Another study reported that canon-ical Wnt signaling is activated in therapy-induced senescence of


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cancer cells and can enhance the tumor initiation capacity ofcancer cells released or escaping from senescence (Milanovicet al., 2018). In addition, blockade of Wnt signaling by smallmolecular inhibitors or antibodies targetingFrizzleds orRSPOscan significantly enhance the toxicity of chemotherapy orradiotherapy or resensitize the resistant tumors to thetreatment inmany studies. Notably, except for thoseWnt-driventumors, anti–Wnt signaling treatment itself normally doesnot show potent suppressive effect on tumor growth but cansignificantly enhance the therapeutic effects of conventionalchemotherapeutic agents in combinational treatment, furthersuggesting that Wnt signaling has a specific role in mediatingresistance to chemotherapy (Nagaraj et al., 2015; Chartieret al., 2016; Fischer et al., 2017a,b; Trillsch et al., 2017).Wnt Signaling Mediated Resistance to Targeted

Therapy. Wnt signaling also confers resistance againstmore targeted therapies. Prostate cancer is hormone sensi-tive; therefore, most patients with prostate cancer who cannotbe cured by surgery or radiation undergo androgen depri-vation therapy. However, the majority of those patientseventually develop lethal metastatic castration–resistantprostate cancer. Some of the resistance is due to intracrinebiosynthesis of androgens, which can be blocked by abir-aterone acetate/prednisone (AA/P). But there are still manypatients who fail to respond to AA/P. A recent clinical studyshowed that the AA/P nonresponders have more alterationsof Wnt pathway components in their tumors, resulting inelevated Wnt/b-catenin signaling. Organoids derived fromtumors of the nonresponders are also resistant to AA/P butcan be resensitized to AA/P by Wnt pathway inhibition (Wanget al., 2018). This finding is consistent with preclinical studyresults that show that Wnt/b-catenin signaling can be acti-vated in prostate cancer cells after androgen deprivation topromote androgen-independent growth, and combining antian-drogenagentwith aWntpathway inhibitor can achieve enhancedgrowth suppression effect in both androgen-dependent and-independent prostate cancer cells (Wang et al., 2008; Leeet al., 2015). In addition to the canonical Wnt/b-cateninsignaling, it is also reported that the noncanonicalWnt signalingis activated by WNT5A and WNT7B in the circulating tumorcells, which mediates resistance to the androgen receptorinhibitor (Miyamoto et al., 2015). Finally, both canonical andnoncanonical Wnt signaling are involved in the bone metas-tases that are common in prostate cancer (Dai et al., 2008;Nandana et al., 2017).Active Wnt signaling also contributes to resistance to growth

factor signaling pathway inhibitors. A high nuclear b-cateninlevel is associated with resistance to PI3K/AKT/mTOR inhib-itors in patients with colorectal cancer (Arqués et al., 2016).Mechanistically, inhibition of PI3K/AKT signaling promotesnuclear accumulation of the proapoptotic tumor suppressorFOXO3a. However, high nuclear b-catenin confers resistanceto the FOXO3a-mediated apoptosis and subverts FOXO3a topromote metastasis in colon cancer, which can be reversed bytankyrase inhibition (Tenbaum et al., 2012; Arqués et al.,2016). In preclinical studies, elevated Wnt signaling has beenobserved in PI3K inhibitor–treated breast cancer cells andBRAF inhibitor–treated colorectal cancer cells. PI3K inhibi-tion promotes the expression of Wnt ligands in breast cancers,whereas BRAF inhibition upregulates b-catenin signalingthrough activating the focal adhesion kinase in colorectalcancer. Blocking Wnt/b-catenin signaling by tankyrase inhibitor

or PORCN inhibitor can significantly enhance the tumor sup-pression effect of these small molecular inhibitors targeting thegrowth factor signaling pathways (Tzeng et al., 2015; Solzaket al., 2017; Chen et al., 2018; Solberg et al., 2018). In EGFR-mutated lung cancer, even though EGFR inhibitors are notobserved to significantly upregulate Wnt signaling in preclinicalstudies, Wnt/b-catenin signaling has been shown to driveresistance to EGFR inhibitors in lung cancer cells becausetankyrase inhibitor and EGFR inhibitor in combination cansynergistically suppress lung cancer cell growth in vitro andin vivo (Casás-Selves et al., 2012; Scarborough et al., 2017).Bromodomain and extra terminal protein (BET) inhibitors

are first-in-class targeted therapies that entered clinical trialsfor treating acute myeloid leukemia. Two independent studiesfound that Wnt/b-catenin signaling promotes primary andacquired resistance to BET inhibition in leukemia (Fong et al.,2015; Rathert et al., 2015). Mechanistically, BET inhibitorsdisrupt the BRD4-chromatin interaction and repress BRD4-dependent transcription of genes such as MYC to suppresscancer progression. However, in the presence of BET inhibitor,b-catenin binds to the promoter regions that are originallyoccupied by BRD4, and maintains the expression levels ofkey target genes including MYC. This evidence establishesWnt/b-catenin signaling as a major driver and candidatebiomarker of resistance to BET inhibitors in a subpopulationof leukemia cells that show high transcriptional plasticity.Recently, two independent studies revealed that Wnt

signaling mediates resistance to complete response to Hedgehogsignaling inhibition in basal cell carcinoma (BCC) (Biehs et al.,2018; Sánchez-Danés et al., 2018). BCC is the most commonmalignancy in humans and typically arises due to constitutivelyactivation of Hedgehog signaling. Treatment with Hedgehogsignaling inhibitor vismodegib mediates differentiation of BCCcells from a hair follicle stem cell like phenotype to an inter-follicular epidermis or isthmus cell fate, and thereby leads totumor regression (Sánchez-Danés et al., 2018). However, BCCtumors always relapse after vismodegib withdrawal (Tanget al., 2016). Itwas found that a small population of LGR51BCCcells with active Wnt signaling escape from vismodegib-induceddifferentiation and mediate tumor relapse after treatment dis-continuation. Importantly, combining vismodegib with PORCNinhibitor led to the eradication of persistent BCC cells andprevented tumor relapse in mouse models.Wnt Signaling Mediated Resistance to Immunother-

apy. The roles of Wnt signaling in regulating normal hemato-poiesis remains a controversial topic (Staal et al., 2008; Kabiriet al., 2015). However, increasing evidence from recent studiessuggests that Wnt/b-catenin signaling drives the primary,adaptive, and acquired resistance to cancer immunotherapy,first in the anti–CTLA-4/PD-1/PD-L1 treatment of melanomabut in other settings as well (Luke et al., 2019). In humanmetastatic melanoma samples, activation of Wnt/b-cateninsignaling correlates with the absence of a T-cell gene expres-sion signature, which reflects a lack of T-cell infiltration.Activation of tumor intrinsic b-catenin signaling suppressesthe expression of tumor-cell–intrinsic chemokines such as CCL4,thereby suppressing the recruitment of CD1031 dendriticcells and subsequent activation of CD81 T-cells (Sprangeret al., 2015). The melanoma derived WNT5A can also activateb-catenin signaling in the dendritic cells in a paracrine mode,which upregulates the expression and activity of the indole-amine 2,3-dioxygenase-1 enzyme in local dendritic cells.


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This promotes the development of tolerogenic dendritic cellsand drives the differentiation of regulatory T-cells (Holtzhausenet al., 2015; Zhao et al., 2018). Concurrent inhibition ofWnt/b-catenin signaling enhanced the effect of immunecheckpoint inhibitors in select preclinical models.Anti-Wnt Signaling–Based Combination Cancer Thera-

pies. As discussed previously, Wnt signaling mediates re-sistance to several therapeutic modalities in clinical practiceas well as multiple anti-cancer agents developed in preclinicalstudies. Therefore, several drug combination studies combin-ingWnt-signaling inhibitors with other conventional cytotoxicagents or targeted inhibitors have been tested in preclinicalstudies and shown improved efficacy. Some of them haveadvanced into clinical trials. Moreover, recent technicaladvances in genome sequencing and genetic screening identi-fied a group of genetically defined cancers that are addicted toWnt signaling and other oncogenic pathways, such as a subsetof colon cancers that harbor concurrent mutations in BRAFand RNF43 or RSPOs (Yan et al., 2017) and RNF43-mutantpancreatic cancers that showhigh dependency onPI3K/mTORsignaling (Zhong et al., 2019). These findings inspired thedevelopment of PORCN inhibitor and growth factor pathwayinhibitor combinational treatment in preclinical studies andclinical trials. Here, we summarize the drug combinationswith Wnt pathway inhibitors for cancer therapy in bothpreclinical studies and clinical trials (Table 3).

Resistance to Wnt Pathway Blockade in NormalTissue and Wnt-Driven Cancer

As discussed previously, dysregulated Wnt signaling is thedriver of a series of genetically defined cancers, and elevatedWnt signaling can mediate resistance to various therapeuticstrategies. Therefore, targeting Wnt-signaling pathways hasbeen attracting attention in the field of cancer research fora long time. Researchers have developed different strategiesto block Wnt signaling, including antibodies targeting recep-tors or agonists, PORCN inhibitors, tankyrase inhibitors, etc.These strategies may apply to different cases based on thespecific alterations in the Wnt pathway. However, becauseWnt signaling also maintains the homeostasis of normaltissue including bone, taste buds, and hair and gut stemcells, one common concern in using Wnt pathway inhibitorsis the potential adverse effect on normal tissues with highdependency on Wnt signaling, such as the gastrointestinaltract. Indeed, tankyrase inhibitors have been shown to causesevere toxicity to the gut as well as loss of body weight in micewhen used for cancer treatment, which limits the use oftankyrase inhibitors in human patients (Lau et al., 2013).However, surprisingly, PORCN inhibitors can be well toler-ated by mice at therapeutic doses for cancer but showing nosignificant side effect on gut (Liu et al., 2013; Proffitt et al.,2013; Madan et al., 2016). In cancers, even those driven byRNF43 mutations can develop resistance to Wnt blockadebecause two pancreatic cancer cell lines with the same RNF43loss-of-functionmutation show different sensitivity to PORCNinhibitor. Interestingly, they were isolated from the same metas-tasis of the same patient, but one cell line is sensitive, whereasthe other is resistant to PORCN inhibitor (Jiang et al., 2013).In colorectal cancers with APC mutations, some cell lines aresensitive to tankyrase inhibitors, whereas others are resis-tant, even though the expression of Wnt target genes can also

be repressed by tankyrase inhibitors in the resistant cell lines(Lau et al., 2013). This observation reveals that there arepotential mechanisms that mediates resistance to Wnt path-way inhibitors in normal tissues and cancers. Three mecha-nisms are described here to explain the resistance.ABC Transporters. ABC transporters are members of

a transport system superfamily. The ABC transporters consistof two ATP-binding domains and two transmembrane domains.These four domains can present in one peptide as a protein ortwo peptides that form a dimer. The two transmembranedomains typically contain twelve a-helices that form a pore-likestructure across the membrane. The ATP-binding domainsthat have ATPase activity use the energy of ATP binding andhydrolysis to drive the conformational change of the trans-membrane domains, thereby energizing the translocation ofsubstrates across the membrane. There are many members inthe ABC transporter family that are responsible for uptake orexport of various substrates for the cell (Glavinas et al., 2004).ABC transporters are known to cause the MDR phenotype

in cancer cells by pumping diverse anticancer drugs out of thecell. ABCB1 is the most extensively studied ABC transporterrelated to drug efflux and is also called MDR1 (Robey et al.,2018). Many studies have shown that the expression of ABCtransporters is upregulated when tumor cells get resistant tocertain drugs, and this always results inmultidrug resistance.So far, there is no direct evidence to show that resistanceto Wnt pathway inhibitors is because of ABC transporters.However, one recent study from our group shows that inthe mouse intestine, the stroma cells that supply Wnts andRSPOs and form the niche for the intestinal stem cells expressa subset of ABC transporters, and are therefore resistant tovarious xenobiotics, including PORCN inhibitors (Chee et al.,2018). This property of the intestinal stroma cells could be dueto the unpredictable environment rich in various xenobioticsin thegastrointestinal tract.But it canwell explain thedifferentialtoxicities that tankyrase inhibitors and PORCN inhibitors haveon the gut, as tankyrase inhibitors target theWnt responding cells(the stem cells) directly, whereas PORCN inhibitors target theWnt suppling cells, the resistant stroma cells here. This findingprovides the rationale for the safety of the gutwhenusingPORCNinhibitors in cancer therapy.Further Mutations of Wnt Pathway Components. It

is generally accepted that upstream Wnt pathway inhibitorssuch as PORCN inhibitors are less effective in cancer cells withmutations of downstream Wnt pathway components such asAPCmutations, even though it is also reported thatWnt ligandsare required to maintain the high levels of Wnt signaling incolorectal cancer cells with APCmutations (Voloshanenko et al.,2013). Besides, it has been observed in many cancer types thatinactivatingmutations ofRNF43/ZNRF3,RSPOs translocation,activating mutations of b-catenin, and loss of APC are almostmutually exclusive, which reflects a rule in tumorigenesis thatWnt/b-catenin signaling only need to be activated in one way oranother. However, co-occurrence of mutations in both upstreamand downstream Wnt pathway does exist in some rare cases,in which the PORCN inhibitors may not work even thoughalterations of RNF43/ZNRF3 or RSPOs can be detected. Inaddition, mutations in downstream components of the Wntpathway can happen during the long-term treatment withupstream Wnt pathway inhibitors. These resistant clonesmay be selected by the treatment and become the dominantpopulation of the tumor. This process has been reported in


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TABLE 3Anti-Wnt signaling-based drug combinations in cancer therapy

Drug A Drug B (Wnt PathwayInhibitor) Drug Combination Rationales/Preclinical Results Clinical Trials

Paclitaxel and carboplatin(chemo agents)

Ipafricept/OMP-54F28(decoy Wnt recptor)

The drug combinations showed synergistic andpotent tumor growth inhibition in patient-derived ovarian, pancreatic, hepatocellular,breast, lung, and/or colorectal cancerxenografts (Chartier et al., 2016; Fischeret al., 2017a,b; Jimeno et al., 2017).

Phase 1 clinical trial (NCT02092363)in patients with recurrentplatinum-sensitive ovarian cancer

Nab-paclitaxel and gemcitabine(chemo agents)

Phase 1 clinical trial (NCT02050178)in patients with previouslyuntreated stage IV pancreaticcancer

Sorafenib (RAF, VEGFR, andPDGFR inhibitor)

Phase 1 clinical trial (NCT02069145)in patients with hepatocellularcancer

Paclitaxel (chemo agent) Vantictumab/OMP-18R5 (anti-Frizzledsantibody)

Phase 1 clinical trial (NCT01973309)in patients with locally recurrent ormetastatic breast cancer

Docetaxel (chemo agents) Phase 1 clinical trial (NCT01957007)in patients with previously treatednon-small-cell lung carcinoma

Nab-paclitaxel and gemcitabine(chemo agents)

Phase 1 clinical trial (NCT02005315)in patients with previouslyuntreated stage IV pancreaticcancer

Taxol, gemcitabine, or irinotecan(chemo agents)

Anti-RSPO1, anti-RSPO2, and anti-RSPO3 antibodies

FOLFIRI (chemo agents) OMP-131R10 (anti-RSPO3 antibody)

Phase 1 clinical trial (NCT02482441)in patients with metastaticcolorectal cancer

API2 (AKT inhibitor) NVP-TNKS656(tankyraseinhibitor)

Tankyrase inhibition reverted resistance toAKT inhibition in patient-derived colorectalcancer xenografts (Arques et al., 2016).

Gefitinib or AZD9291 (EGFRinhibitors)

AZ1366 (tankyraseinhibitor)

The drug combination significantly sloweddown growth of non–small cell lung cancerorthotopic xenografts and improved survivalof tumor-bearing mice (Scarborough et al.,2017).

Vemurafenib (BRAF inhibitor) ICG-001 (blockingb-catenin/TCFinteraction)

BRAF inhibition upregulated b-cateninsignaling in colorectal cancer cell linesin vitro. The drug combination led tosynergistic tumor growth inhibition in HT29xenografts (Chen et al., 2018).

LGX818 (BRAF inhibitor) andCetuximab (EGFR inhibitor)

LGK974 (PORCNinhibitor)

A subset of colorectal cancers harborconcurrent mutations in BRAF and RNF43or RSPOs (Yan et al., 2017).

Phase 1/2 clinical trial(NCT02278133) in patients withBRAF-mutant metastatic colorectalcancer with Wnt pathwaymutations

Buparlisib/BKM120 (pan-PI3Kinhibitor)

LGK974 (PORCNinhibitor)

Burparlisib treatment upregulated Wntsignaling in triple negative breast cancer(TNBC) cell lines in vitro. The drugcombination led to synergistic tumor growthinhibition in xenografts of TNBC cell lineTMD231 (Solzak et al., 2017).

Multiple PI3K/mTOR inhibitorsincluding GDC-0941,ZSTK474, LY-294002,AS252424, and Ku0063794

ETC-159 (PORCNinhibitor)

ETC-159 in combination with one of the fivePI3K/mTOR inhibitors synergisticallysuppressed 3D colony formation of severalWnt-addicted pancreatic andcholangiocarcinoma cell lines. ETC-159 incombination with GDC-0941 led tosynergistic tumor growth inhibition inHPAF-II and AsPC-1 xenografts (Zhonget al., 2019).

Olaparib (PARP inhibitor) Pyrvinium (CK1aactivator) orXAV939 (tankyraseinhibitor)

Wnt/b-catenin signaling mediated resistanceto PARP inhibition in ovarian cancer due inpart to upregulation of DNA damage repair.Inhibition of b-catenin signaling revertedresistance to olaparib in ovarian cancerxenografts (Fukumoto et al., 2019;Yamamoto et al., 2019).

Vismodegib (Hedgehog signalinginhibitor)

LGK974 (PORCNinhibitor) or anti-LRP6 antibody

A LGR51 tumor cell population maintained byactive Wnt signaling persists vismodegibtreatment in human and mouse basal cellcarcinoma and mediates relapse aftertreatment discontinuation. The drugcombination reduced tumor burden

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a colorectal cancer cell line VACO6. VACO6 harbors an RSPO3translocation and is sensitive to the PORCN inhibitor LGK974.However, these cells developed resistance to LGK974 within3 months when cultured with a sublethal concentration ofLGK974. Exome sequencing of the resistant VACO6 clonefound truncating mutations in AXIN1 that are absent inthe parental cell line (Picco et al., 2017). Interestingly,these truncating mutations have been reported in the Catalogueof Somatic Mutations in Cancer database as cancer-relatedsomatic variants. This study demonstrates that further muta-tions in downstream components of Wnt signaling can confersecondary resistance to genetically defined cancers that aresensitive to upstream Wnt pathway blockade.Activation of Other Compensatory Signaling Pathways.

Wnt signaling can mediate resistance to inhibitors of othersignaling pathways, especially the growth factor signalingpathways, and so it is not surprising that activation of thesegrowth factor signaling pathways can compensate for Wntpathway blockade. For example, mTOR signaling is reportedto mediate resistance to tankyrase inhibitors in colorectal cancer(Mashimaet al., 2017).Mechanistically,most of the pro-oncogenicand anti-oncogenic targets are regulated by more than onepathway. For example, c-Myc, as a Wnt-signaling effector, isregulated at the transcriptional level and post-translationallevel by Wnt signaling, as well as PI3K/AKT/mTOR signaling,MAPK signaling, etc. Therefore, inhibiting one signaling inputcan be compensated by other regulators and may have aminimal effect on the output. Notably, in many Wnt-drivencancers, the growth factor signaling pathways are alsohyperactivated due to mutations in EGFR, KRAS, BRAF,PI3K, PTEN, etc. Therefore, developing combination drugtherapy is a promising solution to maximize the therapeuticeffects.

Concluding RemarksSince the discovery of the first mammalianWnt gene and its

function in the mouse mammary cancer nearly four decadesago, the Wnt-signaling pathway has been extensively inves-tigated worldwide. Although these studies comprehensivelyrevealed the molecular mechanisms and functional effects ofthe Wnt signaling in normal status and diseases, numerousquestions still remain to be answered. Recent findings inthe Wnt field continue to extend our understanding of thisevolutionarily conservedancient signalingpathway.Asahigh-light of this review, an increasing number of studies revealand confirm the key roles ofWnt signaling in cancer initiation,

progression, and drug resistance, inspiring pharmacologicaltargeting of Wnt signaling in patients.Importantly, increasing knowledge in the Wnt field have

facilitated the development of multiple Wnt pathway inhib-itors along with rational drug combination strategies. Theseinhibitors and drug combinations showed promising efficacyin carefully selected preclinical cancer models, and some haveadvanced to clinical trials. However, considering the impor-tant roles of Wnt signaling in maintaining normal tissuehomeostasis, the on-target adverse effects of Wnt pathwayinhibitors cannot be neglected. Notably, although thoseupstream Wnt pathway inhibitors including PORCN inhibitorsand the anti-Frizzleds antibody advanced to clinical trials andshowed manageable side effects, several of the downstreaminhibitors such as the tankyrase inhibitors have remained at thepreclinical stage, possibly due to the toxicity to the gastrointes-tinal tract. Therefore, it will be important to both develop moreselective inhibitors that have minimal toxicity on normaltissue and also develop synergistic drug combinations so thatlower dosages can be used to improve both the therapeuticindex and the anticancer efficacy.

Acknowledgments

We acknowledge Sivakumar Parthiban for assistance with theconsensus modeling of Wnt proteins. Z.Z. acknowledges ShazibPervaiz for the supervision on his training. We thank all membersof the Virshup laboratory for useful discussions on this work andapologize to our colleagues whose work could not be cited here dueto space limitation.

Authorship Contributions

Performed data analysis: Zhong, Virshup.Wrote or contributed to the writing of the manuscript: Zhong,

Virshup.

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TABLE 3—Continued

Drug A Drug B (Wnt PathwayInhibitor) Drug Combination Rationales/Preclinical Results Clinical Trials

(vismodegib 1 anti-LRP6 antibody) oreliminated resistant tumor cells (vismodegib1 LGK974) in mouse models (Biehs et al.,2018; SanchezDanes et al., 2018).

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Preclinical studies showed that Wnt signalingmediated resistance to immunotherapies,and concurrent inhibition of Wnt/b-cateninsignaling enhanced the efficacy of immunecheckpoint inhibitors in melanoma mousemodels (Holtzhausen et al., 2015; Sprangeret al., 2015; Zhao et al., 2018).

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Address correspondence to: David M. Virshup, Program in Cancer andStem Cell Biology, Duke-NUS Medical School, 8 College Road, Singapore169857, Singapore. E-mail: [email protected]


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